This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glioblastoma (GBM), the most common type of primary brain tumor, is an aggressive, highly invasive and neurologically destructive tumor considered to be among the deadliest of all human cancers. While it is known that GBM are tumors of glial origin, a continuing and intense debate surrounds whether they arise from differentiated astrocytes, committed astrocyte progenitors or undifferentiated neural stem cells (NSC). My laboratory has focused on the development of spontaneous genetically engineered mouse (GEM) models of glioblastoma using astrocyte- and NSC- specific promoters that I identified through a comprehensive gene expression analysis of astrocyte and NSC development in normal mouse brain. Using a new generation of transgenic mice that our laboratory has developed, we can now target oncogenes and tumor suppressor genes known to be important in glioblastoma, to either the NSC or astrocyte compartment in the adult mouse brain. The second mouse model system that we are using for our studies is our newly developed orthotopic model using glioblastoma tumor taken from a patient at the time of initial surgical resection and, after minimal dissociation, injected into SCID mouse brains. This model is called a human orthotopic glioblastoma model. When the human tumor grows to approximately 1500 mg in the mouse brain, which fills most of one hemisphere, we infuse 13C-glucose and subsequently sacrifice the animal and take the tumor for NMR spectral analysis. We have generated tumor lines from over 40 patients and these represent a comprehensive sampling of the genetic mutations that characterize glioblastoma. Thus, we can compare the metabolic profiles of the various subgroups of mutational profiles (for example, EGFR amplified, PTEN deleted vs. EGFR normal copy number, PTEN deleted). Using this combination of mouse model and NMR 13C spectral analysis, we have made observations in vivo that challenge the longstanding dogma first described by Warburg over 60 years ago. The objectives of our ongoing collaboration with the investigators in the Research Resource are as follows: (i) To define the metabolic profile of primary NSC and astrocytes isolated from the GEM mice and de novo tumors that arise from these respective cell types after switching on the most important GBM relevant mutations. (ii) We will also undertake cross species analysis comparing patterns of glucose metabolism of de novo mouse gliomas (GEM) and glucose utilization by primary human GBM tumors in the orthotopic tumor model. The goal of this comparison is to test whether the metabolic prolife of NSC or astrocyte-derived mouse tumors more closely mimics that of human orthotopic tumors. (iii) We will develop the technology to be able to use small 20 mg) tissue extracts in these studies. When optimized, we will be able to study metabolism in the early stages of tumor development and to compare regions of the tumor that are thought to be heterogeneous with respect to oxygen content or gene expression. This will be particularly useful for the study of brain metastases where we will be able to study individual tumors of small size and compare their metabolic profiles to the spontaneous gliomas and human orthotopic tumors. There are three main projects that are in various stages of completion that address these objectives. Project1: To measure the relative rates of glucose and acetate oxidation, glycolysis to lactate, and the pentose phosphate pathway in murine glioblastoma derived from NSC and astrocytes. Tumor metabolism will be assessed by infusion of [U-13C]glucose plus [1,2-13C] acetate in conscious mice which have been confirmed by MRI to have a contrast enhancing mass. Since acetate is only oxidized by astrocytes, it is expected that astrocyte derived tumors will be labeled by glutamate or glutamine in position 4 but not 5. If NSC derived tumors show a similar metabolic profile, it will strongly support that notion that, irrespective of the cell of origin, transformed glioma cells share a common metabolic profile. It is also possible that NSC derived tumors have a distinctly different metabolic profile, possibly one that resembles normal NSC. Such information would also be of significant interest to the larger neuroscience community. Project 2: To examine intermediary metabolism in brain metastases using the human orthotopic tumor model and determine the variation in pathway activity based on histological subtype. The four most common types of brain metastases will be used for these studies. These include non-small cell lung cancer, breast, renal cell, melanoma. Project 3: To compare and contrast the metabolic phenotype of brain metastases with glioblastoma and determine the impact of the underlying status of the common cancer pathways, RAS-MAPK, AKT, and p53 pathways relative to the underlying histology. We will determine whether the genotype or the cell of origin is more important in determining the metabolic phenotype.